RESUMEN
BACKGROUND: Acute kidney injury (AKI) is associated with a severe decline in kidney function caused by abnormalities within the podocytes' glomerular matrix. Recently, AKI has been linked to alterations in glycolysis and the activity of glycolytic enzymes, including pyruvate kinase M2 (PKM2). However, the contribution of this enzyme to AKI remains largely unexplored. METHODS: Cre-loxP technology was used to examine the effects of PKM2 specific deletion in podocytes on the activation status of key signaling pathways involved in the pathophysiology of AKI by lipopolysaccharides (LPS). In addition, we used lentiviral shRNA to generate murine podocytes deficient in PKM2 and investigated the molecular mechanisms mediating PKM2 actions in vitro. RESULTS: Specific PKM2 deletion in podocytes ameliorated LPS-induced protein excretion and alleviated LPS-induced alterations in blood urea nitrogen and serum albumin levels. In addition, PKM2 deletion in podocytes alleviated LPS-induced structural and morphological alterations to the tubules and to the brush borders. At the molecular level, PKM2 deficiency in podocytes suppressed LPS-induced inflammation and apoptosis. In vitro, PKM2 knockdown in murine podocytes diminished LPS-induced apoptosis. These effects were concomitant with a reduction in LPS-induced activation of ß-catenin and the loss of Wilms' Tumor 1 (WT1) and nephrin. Notably, the overexpression of a constitutively active mutant of ß-catenin abolished the protective effect of PKM2 knockdown. Conversely, PKM2 knockdown cells reconstituted with the phosphotyrosine binding-deficient PKM2 mutant (K433E) recapitulated the effect of PKM2 depletion on LPS-induced apoptosis, ß-catenin activation, and reduction in WT1 expression. CONCLUSIONS: Taken together, our data demonstrates that PKM2 plays a key role in podocyte injury and suggests that targetting PKM2 in podocytes could serve as a promising therapeutic strategy for AKI. TRIAL REGISTRATION: Not applicable. Video abstract.
Asunto(s)
Lesión Renal Aguda , Leucemia Mieloide Aguda , Podocitos , Lesión Renal Aguda/metabolismo , Animales , Leucemia Mieloide Aguda/metabolismo , Lipopolisacáridos/farmacología , Ratones , Piruvato Quinasa/genética , Piruvato Quinasa/metabolismo , Piruvato Quinasa/farmacología , beta Catenina/metabolismoRESUMEN
BACKGROUND: Due to a liberalisation reform in 2009, the availability of over-the-counter (OTC) analgesics has increased significantly in the Swedish market over the past decade. With the increasing number of generic products available on the market and the possibility of buying OTC drugs from non-pharmacy outlets, a key to safe drug use is that consumers possess the necessary knowledge to differentiate between the different brands and choose the appropriate drug for their ailments. The aim of this study was to investigate Swedish consumers' knowledge of and attitudes towards generic OTC analgesics. METHODS: A sample of 209 Swedish adults (66% women; mean age 43.1 years) who bought OTC analgesics at a community pharmacy in one of the country's three largest cities responded to a structured questionnaire. The questions related to knowledge of active substances, the use and choice of OTC analgesics (generic or original brand), attitudes towards generic OTC analgesics, information received about OTC analgesics and experience with generic substitution of prescription drugs. RESULTS: Almost one in five reported weekly use of OTC analgesics, and 32% assigned minimum three out of four active substances to the correct brand(s) of OTC analgesics. Among the 50 participants (24%) who assigned all four active substances correctly, it was predominantly women and participants with higher education. Four out of five participants were positive towards the cheaper brands, and 69% reportedly chose cheaper generic brands over more expensive brands. Knowledge about the active substances of different brands was associated with positive attitudes towards generic products. CONCLUSION: Swedish pharmacy customers have to a varying extent the knowledge to differentiate between different brands of commonly used OTC analgesics in terms of active substances. There is a predominantly positive attitude towards generic OTC analgesics, although some consider generic drugs to be inferior and stay loyal to the original brands.
RESUMEN
BACKGROUND: Current pharmacological therapies and treatments targeting pancreatic neuroendocrine tumors (PNETs) have proven ineffective, far too often. Therefore, there is an urgent need for alternative therapeutic approaches. Zyflamend, a combination of anti-inflammatory herbal extracts, that has proven to be effective in various in vitro and in vivo cancer platforms, shows promise. However, its effects on pancreatic cancer, in particular, remain largely unexplored. METHODS: In the current study, we investigated the effects of Zyflamend on the survival of beta-TC-6 pancreatic insulinoma cells (ß-TC6) and conducted a detailed analysis of the underlying molecular mechanisms. RESULTS: Herein, we demonstrate that Zyflamend treatment decreased cell proliferation in a dose-dependent manner, concomitant with increased apoptotic cell death and cell cycle arrest at the G2/M phase. At the molecular level, treatment with Zyflamend led to the induction of ER stress, autophagy, and the activation of c-Jun N-terminal kinase (JNK) pathway. Notably, pharmacological inhibition of JNK abrogated the pro-apoptotic effects of Zyflamend. Furthermore, Zyflamend exacerbated the effects of streptozotocin and adriamycin-induced ER stress, autophagy, and apoptosis. CONCLUSION: The current study identifies Zyflamend as a potential novel adjuvant in the treatment of pancreatic cancer via modulation of the JNK pathway. Video abstract.
Asunto(s)
Apoptosis , Sistema de Señalización de MAP Quinasas , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/patología , Extractos Vegetales/farmacología , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxorrubicina/farmacología , Estrés del Retículo Endoplásmico/efectos de los fármacos , Inflamación/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Modelos Biológicos , Ratas , Estreptozocina/farmacologíaRESUMEN
The prevalence of obesity and its comorbidities has sparked a worldwide concern to address rates of adipose tissue accrual. Recent studies have demonstrated a novel role of Zyflamend, a blend of natural herbal extracts, in regulating lipid metabolism in several cancer cell lines through the activation of the AMPK signalling pathway. Yet, the role of Zyflamend in adipogenic differentiation and lipid metabolism remains largely unexplored. The objective of this study is to investigate the effects of Zyflamend on white 3T3-MBX pre-adipocyte differentiation and elucidate the molecular mechanisms. We demonstrate that Zyflamend treatment altered cell cycle progression, attenuated proliferation, and increased cell death of 3T3-MBX pre-adipocytes. In addition, treatment with Zyflamend inhibited lipid accumulation during the differentiation of 3T3-MBX cells, consistent with decreased expression of lipogenic genes and increased lipolysis. Mechanistically, Zyflamend-induced alterations in adipogenesis were mediated, at least in part, through the activation of AMPK, PKA, and JNK. Inhibition of AMPK partially reversed Zyflamend-induced inhibition of differentiation, whereas the inhibition of either JNK or PKA fully restored adipocyte differentiation and decreased lipolysis. Taken together, the present study demonstrates that Zyflamend, as a novel anti-adipogenic bioactive mix, inhibits adipocyte differentiation through the activation of the PKA and JNK pathways. ABBREVIATION: 7-AAD: 7-amino-actinomycin D; ACC: acetyl-CoA carboxylase; AKT: protein kinase B; AMPK: AMP-activated protein kinase; ATGL: adipose triglyceride lipase; C/EBPα: CCAAT-enhancer binding protein alpha; DMEM: Dulbecco's Modified Eagle Medium; DMSO: dimethyl sulphoxide; DTT: dithiothreitol; EGTA: ethylene glycol-bis-(2-aminoethyl)-N,N,N',N'-tetraacetic acid; ERK: extracellular signal-regulated kinases; FASN: fatty acid synthase; FBS: foetal bovine serum; GLUT: glucose transporter; HSL: hormone-sensitive lipase; IR: insulin receptor; IRS: insulin receptor substrate; JNK: c-JUN N-terminal kinase; MGL: monoacylglycerol lipase; NaF: sodium fluoride; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; PBS: phosphate buffered- saline; PCB: pyruvate carboxylase; PDE: phosphodiesterase; PKA: protein kinase cAMP-dependent; PMSF: phenylmethylsulfonyl fluoride; PPARγ: perilipin peroxisome proliferator-activated receptor gamma; PREF-1: pre-adipocyte factor 1; PVDF: polyvinylidene fluoride; RIPA: radio-immunoprecipitation assay; SDS-PAGE: sodium dodecyl sulphate polyacrylamide gel electrophoresis; SEM: standard error of the mean; SOX9: suppressor of cytokine signalling 9; TGs: triacylglycerols.
Asunto(s)
Adipogénesis/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Extractos Vegetales/farmacología , Transducción de Señal/efectos de los fármacos , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Puntos de Control del Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Lipólisis , RatonesRESUMEN
Pyruvate kinase M2 is a critical enzyme that regulates cell metabolism and growth under different physiological conditions. In its metabolic role, pyruvate kinase M2 catalyzes the last glycolytic step which converts phosphoenolpyruvate to pyruvate with the generation of ATP. Beyond this metabolic role in glycolysis, PKM2 regulates gene expression in the nucleus, phosphorylates several essential proteins that regulate major cell signaling pathways, and contribute to the redox homeostasis of cancer cells. The expression of PKM2 has been demonstrated to be significantly elevated in several types of cancer, and the overall inflammatory response. The unusual pattern of PKM2 expression inspired scientists to investigate the unrevealed functions of PKM2 and the therapeutic potential of targeting PKM2 in cancer and other disorders. Therefore, the purpose of this review is to discuss the mechanistic and therapeutic potential of targeting PKM2 with the focus on cancer metabolism, redox homeostasis, inflammation, and metabolic disorders. This review highlights and provides insight into the metabolic and non-metabolic functions of PKM2 and its relevant association with health and disease.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica , Inflamación/enzimología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Enfermedades Metabólicas/enzimología , Hormonas Tiroideas/metabolismo , Adenosina Trifosfato/metabolismo , Aterosclerosis/enzimología , Proliferación Celular , Glucólisis , Homeostasis , Humanos , Enfermedades Inflamatorias del Intestino/enzimología , Insulina/metabolismo , Enfermedades Renales/enzimología , Hígado/enzimología , Naftoquinonas/farmacología , Metástasis de la Neoplasia , Neoplasias/enzimología , Neuralgia/enzimología , Oxidantes/metabolismo , Oxidación-Reducción , Isoformas de Proteínas , Sepsis/enzimología , Transducción de Señal , Distribución Tisular , Proteínas de Unión a Hormona TiroideRESUMEN
BACKGROUND: Zyflamend, a blend of herbal extracts, effectively inhibits tumor growth using preclinical models of castrate-resistant prostate cancer mediated in part by 5'-adenosine monophosphate-activated protein kinase (AMPK), a master energy sensor of the cell. Clinically, treatment with Zyflamend and/or metformin (activators of AMPK) had benefits in castrate-resistant prostate cancer patients who no longer responded to treatment. Two predominant upstream kinases are known to activate AMPK: liver kinase B1 (LKB1), a tumor suppressor, and calcium-calmodulin kinase kinase-2 (CaMKK2), a tumor promotor over-expressed in many cancers. The objective was to interrogate how Zyflamend activates AMPK by determining the roles of LKB1 and CaMKK2. METHODS: AMPK activation was determined in CWR22Rv1 cells treated with a variety of inhibitors of LKB1 and CaMKK2 in the presence and absence of Zyflamend, and in LKB1-null HeLa cells that constitutively express CaMKK2, following transfection with wild type LKB1 or catalytically-dead mutants. Upstream regulation by Zyflamend of LKB1 and CaMKK2 was investigated targeting protein kinase C-zeta (PKCζ) and death-associated protein kinase (DAPK), respectively. RESULTS: Zyflamend's activation of AMPK appears to be LKB1 dependent, while simultaneously inhibiting CaMKK2 activity. Zyflamend failed to rescue the activation of AMPK in the presence of pharmacological and molecular inhibitors of LKB1, an effect not observed in the presence of inhibitors of CaMKK2. Using LKB1-null and catalytically-dead LKB1-transfected HeLa cells that constitutively express CaMKK2, ionomycin (activator of CaMKK2) increased phosphorylation of AMPK, but Zyflamend only had an effect in cells transfected with wild type LKB1. Zyflamend appears to inhibit CaMKK2 by DAPK-mediated phosphorylation of CaMKK2 at Ser511, an effect prevented by a DAPK inhibitor. Alternatively, Zyflamend mediates LKB1 activation via increased phosphorylation of PKCζ, where it induced translocation of PKCζ and LKB1 to their respective active compartments in HeLa cells following treatment. Altering the catalytic activity of LKB1 did not alter this translocation. DISCUSSION: Zyflamend's activation of AMPK is mediated by LKB1, possibly via PKCζ, but independent of CaMKK2 by a mechanism that appears to involve DAPK. CONCLUSIONS: Therefore, this is the first evidence that natural products simultaneously and antithetically regulate upstream kinases, known to be involved in cancer, via the activation of AMPK.